Abstract
The formation of disulfides within proteins entering the secretory pathway is catalyzed by the protein disulfide isomerase family of endoplasmic reticulum localized oxidoreductases. One such enzyme, ERp57, is thought to catalyze the isomerization of non-native disulfide bonds formed in glycoproteins with unstructured disulfide-rich domains. Here we investigated the mechanism underlying ERp57 specificity toward glycoprotein substrates and the interdependence of ERp57 and the calnexin cycle for their correct folding. Our results clearly show that ERp57 must be physically associated with the calnexin cycle to catalyze isomerization reactions with most of its substrates. In addition, some glycoproteins only require ERp57 for correct disulfide formation if they enter the calnexin cycle. Hence, the specificity of ER oxidoreductases is not only determined by the physical association of enzyme and substrate but also by accessory factors, such as calnexin and calreticulin in the case of ERp57. These conclusions suggest that the calnexin cycle has evolved with a specialized oxidoreductase to facilitate native disulfide formation in complex glycoproteins.
Highlights
Densitometry scans of the each lane confirmed that a more compact band was observed in the absence of castanospermine (Fig. 6). For this substrate, there is a requirement to engage with the calnexin cycle to ensure correct disulfide formation; blocking such an interaction does not allow correct folding by providing access to other oxidoreductases including non-calnexin-associated ERp57
Substrate specificity is directly related to the sequestration of glycoproteins into the calnexin cycle, but this specificity is not a consequence of a direct interaction of ERp57 with substrates; rather, it is mediated by the presence of a monoglucosylated oligosaccharide side chain
One of the reasons for the proliferation of ER oxidoreductases is the fact that the substrates for disulfide formation can be sequestered into subcomplexes, which require both spatial and temporal coordination with oxidoreductases to catalyze oxidation, isomerization, or reduction
Summary
Antibodies and Cell Lines—Mouse monoclonal antibody (8E3) to 1-integrin was a gift from Martin Humphries (University of Manchester, Manchester, UK). Transcription and translation in semipermeabilized (SP) cells were performed essentially as described previously (13). 1-integrin translation products were immunoisolated prior to electrophoresis; otherwise, SP cells were isolated and resuspended in SDS-PAGE sample buffer (31.25 mM Tris-HCl, pH 6.8, 2% w/v SDS, 5% v/v glycerol, 0.01% w/v bromphenol blue). Electrophoresis and Western Blotting—Samples for SDSPAGE were resuspended in SDS-PAGE sample buffer, and dithiothreitol (50 mM) was added to reduce samples where indicated. Primary antibody incubations were performed for 1 h at 22 °C with 3% milk, whereas secondary antibodies (polyclonal goat anti-rabbit or rabbit anti-mouse immunoglobulins conjugated to horseradish peroxidase (Dako, Ely, UK)) were diluted 1:2000 in Tris/ Tween-buffered saline and again incubated at 22 °C for 1 h. Dithiothreitol was added to 20 mM for 3 min at 30 °C prior to NEM addition. Two-dimensional Gel Electrophoresis—Non-reducing/reducing two-dimensional electrophoresis and substrate identification were performed exactly as described previously (6)
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